Bendamustine pharmaceutical compositions

ABSTRACT

The present invention provides pharmaceutical formulations of lyophilized bendamustine suitable for pharmaceutical use. The present invention further provides methods of producing lyophilized bendamustine. The pharmaceutical formulations can be used for any disease that is sensitive to treatment with bendamustine, such as neoplastic diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/654,898, filed Oct. 18, 2012, which is a continuation of U.S.application Ser. No. 11/330,868, filed Jan. 12, 2006, which claims thebenefit of U.S. Provisional Application No. 60/644,354, filed Jan. 14,2005, the entireties of which are incorporated herein for all purposes.

FIELD OF THE INVENTION

The present invention pertains to the field of pharmaceuticalcompositions for the treatment of various disease states, especiallyneoplastic diseases and autoimmune diseases. Particularly, it relates topharmaceutical formulations comprising nitrogen mustards, particularlythe nitrogen mustard bendamustine, e.g., bendamustine HCl.

BACKGROUND OF THE INVENTION

The present invention claims the benefit of and priority to U.S. Ser.No. 60/644,354, filed Jan. 14, 2005, entitled, “BendamustinePharmaceutical Compositions,” which is incorporated herein by referencein its entirety, including figures and claims.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that anysuch information is prior art, or relevant, to the presently claimedinventions, or that any publication specifically or implicitlyreferenced is prior art.

Because of their high reactivity in aqueous solutions, nitrogen mustardsare difficult to formulate as pharmaceuticals and are often supplied foradministration in a lyophilized form that requires reconstitution,usually in water, by skilled hospital personal prior to administration.Once in aqueous solution, nitrogen mustards are subject to degradationby hydrolysis, thus, the reconstituted product should be administered toa patient as soon as possible after its reconstitution.

Bendamustine,(4-{5-[Bis(2-chloroethyl)amino]-1-methyl-2-benzimidazolyl}butyric acid,is an atypical structure with a benzimidazole ring, whose structureincludes an active nitrogen mustard (see Formula I, which showsbendamustine hydrochloride).

Bendamustine was initially synthesized in 1963 in the German DemocraticRepublic (GDR) and was available from 1971 to 1992 in that locationunder the name Cytostasan®. Since that time, it has been marketed inGermany under the tradename Ribomustin®. It has been widely used inGermany to treat chronic lymphocytic leukemia, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma, and breast cancer.

Due to its degradation in aqueous solutions (like other nitrogenmustards), bendamustine is supplied as a lyophilized product. Thecurrent lyophilized formulation of bendamustine (Ribomustin®) containsbendamustine hydrochloride and mannitol in a sterile lyophilized form asa white powder for intravenous use following reconstitution. Thefinished lyophilisate is unstable when exposed to light. Therefore, theproduct is stored in brown or amber-colored glass bottles. The currentlyophilized formulation of bendamustine contains degradation productsthat may occur during manufacturing of the drug substance and/or duringthe lyophilization process to make the finished drug product.

Currently bendamustine is formulated as a lyophilized powder forinjection with 100 mg of drug per 50 mL vial or 25 mg of drug per 20 mLvial. The vials are opened and reconstituted as close to the time ofpatient administration as possible. The product is reconstituted with 40mL (for the 100 mg presentation) or 10 mL (for the 25 mg presentation)of Sterile Water for Injection. The reconstituted product is furtherdiluted into 500 mL, q.s., 0.9% Sodium Chloride for Injection. The routeof administration is by intravenous infusion over 30 to 60 minutes.

Following reconstitution with 40 mL Sterile Water for Injection, vialsof bendamustine are stable for a period of 7 hours under roomtemperature storage or for 6 days upon storage at 2-8° C. The 500 mLadmixture solution must be administered to the patient within 7 hours ofvial reconstitution (assuming room temperature storage of theadmixture).

The reconstitution of the present bendamustine lyophilized powder isdifficult. Reports from the clinic indicate that reconstitution canrequire at least fifteen minutes and may require as long as thirtyminutes. Besides being burdensome and time-consuming for the healthcareprofessional responsible for reconstituting the product, the lengthyexposure of bendamustine to water during the reconstitution processincreases the potential for loss of potency and impurity formation dueto the hydrolysis of the product by water.

Thus, a need exists for lyophilized formulations of bendamustine thatare easier to reconstitute and which have a better impurity profile thanthe current lyophilate (lyophilized powder) formulations ofbendamustine.

German (GDR) Patent No. 34727 discloses a method of preparingω-[5-bis-(β-chloroethyl)-amino-benzimidazolyl-(2)]-alkane carboxylicacids substituted in the 1-position.

German (GDR) Patent No. 80967 discloses an injectable preparation ofγ-[1-methyl-5-bis-(β-chloroethyl)-amino-benzimaidazolyl-(2)]-butric acidhydrochloride.

German (GDR) Patent No. 159877 discloses a method for preparing4-[1-methyl-5-bis(2-chloroethyl)amino-benzimidazolyl-2)-butyric acid.

German (GDR) Patent No. 159289 discloses an injectable solution ofbendamustine.

Ribomustin® bendamustine Product monograph (updated January 2002)http://www.ribosepharm.de/pdf/ribomustin_bendamustin/productmonograph.pdfprovides information about Ribomustin® including product description.

Ni et al. report that the nitrosourea SarCNU was more stable in puretertiary butanol than in pure acetic acid, dimethyl sulfoxide,methylhydroxy, water or in TBA/water mixtures (Ni et al. (2001) Intl. J.Pharmaceutics 226:39-46).

Lyophilized cyclophoshamide is known in the art see e.g., U.S. Pat. Nos.5,418,223; 5,413,995; 5,268,368; 5,227,374; 5,130,305; 4,659,699;4,537,883; and 5,066,647.

The lyophilized nitrogen mustard Ifosfamide is disclosed inInternational Publication No. WO 2003/066027; U.S. Pat. Nos. 6,613,927;5,750,131; 5,972,912; 5,227,373; and 5,204,335.

Teagarden et al. disclose lyophilized formulations of prostaglandin E-1made by dissolving PGE-1 in a solution of lactose and tertiary butylalcohol (U.S. Pat. No. 5,770,230).

SUMMARY OF THE INVENTION

The present invention is directed to stable pharmaceutical compositionsof nitrogen mustards, in particular lyophilized bendamustine and its usein treatment of various disease states, especially neoplastic diseasesand autoimmune diseases.

An embodiment of the invention is a pharmaceutical composition ofbendamustine containing not more than about 0.5% to about 0.9% (areapercent of bendamustine) HP1, as shown in Formula II,

at the time of release or where the HP1 is the amount of HP1 present attime zero after reconstitution of a lyophilized pharmaceuticalcomposition of bendamustine as described herein. In a preferredembodiment is a pharmaceutical composition of bendamustine containingnot more than about 0.5% (area percent of bendamustine) HP1, preferablynot more than about 0.45%, more preferably not more than about 0.40%,more preferably not more than about 0.35%, even more preferably not morethan 0.30%.

Another embodiment of the invention is a lyophilized preparation ofbendamustine containing not more than about 0.1% to about 0.3%bendamustine dimer as shown in Formula III at release or at time zeroafter reconstitution

Yet another embodiment of the invention is a lyophilized preparation ofbendamustine containing not more than about 0.5%, preferably 0.15% toabout 0.5%, bendamustine ethylester, as shown in Formula IV at releaseor at time zero after reconstitution

Yet another embodiment of the invention is a lyophilized preparation ofbendamustine wherein the concentration of bendamustine ethylester(Formula IV) is no more than 0.2%, preferably 0.1%, greater than theconcentration of bendamustine ethylester as found in the drug substanceused to make the lyophilized preparation.

In another embodiment of the invention is a lyophilized preparation ofbendamustine containing not more than about 0.5% to about 0.9% (areapercent of bendamustine) HP1 at the time of drug product release. In apreferred embodiment is a lyophilized preparation of bendamustinecontaining not more than about 0.50% (area percent of bendamustine) HP1,preferably not more than about 0.45%, more preferably not more thanabout 0.40%, more preferably not more than about 0.35%, even morepreferably not more than 0.30%. An aspect of this embodiment islyophilized preparations of bendamustine containing not more than about0.5% to about 0.9%, preferably 0.5%, (area percent of bendamustine) HP1at the time of release of drug product where the lyophilized preparationis packaged in a vial or other pharmaceutically acceptable container.

In yet another aspect of the invention, the lyophilized preparations ofbendamustine are stable with respect to the amount of HP1 for at leastabout 6 months, preferably 12 months, preferably 24 months, to about 36months or greater when stored at about 2° to about 30°. Preferredtemperatures for storage are about 5° C. and about room temperature.

Another embodiment of the invention is a pharmaceutical dosage form thatincludes a pharmaceutical composition of bendamustine containing notmore than about 0.5% to about 0.9% HP1, preferably not more than about0.50%, preferably not more than about 0.45%, more preferably not morethan about 0.40%, more preferably not more than about 0.35%, even morepreferably not more than 0.30%, where the HP1 is the amount of HP1present at release or at time zero after reconstitution of a lyophilizedpreparation of bendamustine of the present invention. In preferredaspects of the invention, the dosage form can be about 5 to about 500 mgof bendamustine, about 10 to about 300 mg of bendamustine, about 25 mgof bendamustine, about 100 mg of bendamustine, and about 200 mg ofbendamustine.

Yet another embodiment of the invention is a pharmaceutical dosage formthat includes a lyophilized preparation of bendamustine containing notmore than about 0.5% to about 0.9%, preferably 0.5%, HP1. Preferreddosage forms can be about 5 to about 500 mg of bendamustine, about 10 toabout 300 mg of bendamustine, about 25 mg of bendamustine, about 100 mgof bendamustine, and about 200 mg of bendamustine.

In still another embodiment, the invention includes a pharmaceuticalcomposition of bendamustine including bendamustine containing not morethan about 0.5% to about 0.9% (area percent of bendamustine), preferablynot more than about 0.50%, preferably not more than about 0.45%, morepreferably not more than about 0.40%, more preferably not more thanabout 0.35%, even more preferably not more than 0.30%, and a traceamount of one or more organic solvents, wherein said HP1 is the amountof HP1 present at release or time zero after reconstitution of alyophilized pharmaceutical composition of bendamustine as disclosedherein. In different aspects of this embodiment, the organic solvent isselected from one or more of tertiary butanol, n-propanol, n-butanol,isopropanol, ethanol, methanol, acetone, ethyl acetate, dimethylcarbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methylisobutyl ketone, 1-pentanol, methyl acetate, carbon tetrachloride,dimethyl sulfoxide, hexafluoroacetone, chlorobutanol, dimethyl sulfone,acetic acid, and cyclohexane. Preferred organic solvents include one ormore of ethanol, methanol, propanol, butanol, isopropanol, and tertiarybutanol. A more preferred organic solvent is tertiary butanol, alsoknown as TBA, t-butanol, tert-butyl alcohol or tertiary butyl alcohol.

The present invention involves a method for obtaining agency approvalfor a bendamustine product, the improvement which includes setting arelease specification for bendamustine degradants at less than about4.0%, preferably about 2.0% to about 4.0%, (area percent bendamustine)or otherwise to achieve the pharmaceutical compositions describedherein. An aspect of this embodiment is a method for obtaining agencyapproval for a bendamustine product which includes setting a releasespecification for HP1 to be less than or equal to 1.5% (area percentBendamustine). The bendamustine product herein contains not more thanabout 0.5% (area percent of bendamustine) HP1 at release.

Another embodiment is a method for obtaining agency approval for abendamustine product, the improvement which includes setting ashelf-life specification for bendamustine degradants at less than about7.0%, preferably about 5.0% to about 7.0%, (area percent bendamustine)where the product is stored at about 2° C. to about 30° C. Preferredtemperatures for storage are about 5° C. and about room temperature. Thebendamustine product herein contains not more than about 0.5% (areapercent of bendamustine) HP1 at release.

Another embodiment of the invention is a process for manufacturing alyophilized preparation of bendamustine which includes controlling forthe concentration of bendamustine degradants in the final product, suchthat the concentration of bendamustine degradants is less than about4.0%, preferably no more than about 2.0% to about 4.0%, (area percent ofbendamustine) at release or otherwise to achieve the pharmaceuticalcompositions described herein. The bendamustine product herein containsnot more than about 0.5% to about 0.9%, preferably about 0.5%, (areapercent of bendamustine) HP1 at release.

The present invention discloses a process for manufacturing alyophilized preparation of bendamustine which comprises controlling forthe concentration of bendamustine degradants in the final product, suchthat, at release, the concentration of HP1 is less than 0.9%, preferably0.5%, (area percent of bendamustine) and, at the time of productexpiration, the concentration of bendamustine degradants is less thanabout 7.0%, preferably no more than about 5.0% to about 7.0%; whereinsaid product is stored at about 2° C. to about 30° C.

Another embodiment of the invention is a bendamustine pre-lyophilizationsolution or dispersion comprising one or more organic solvents where thesolution or dispersions include at least one stabilizing concentrationof an organic solvent which reduces the level of degradation ofbendamustine so that the amount of HP1 produced during lyophilizationfrom about 0 to 24 hours does not exceed about 0.5% to about 0.9% (areapercent of bendamustine) preferably 0.50%, preferably 0.45%, morepreferably 0.40%, more preferably 0.35%, even more preferably 0.30%. Anaspect of this embodiment is the lyophilized powder produced from thepre-lyophilization solution or dispersion.

Still another embodiment of the invention is a bendamustinepre-lyophilization solution or dispersion comprising one or more organicsolvents where the solution or dispersions include at least onestabilizing concentration of an organic solvent which reduces the levelof degradation of bendamustine so that the amount of bendamustineethylester produced during lyophilization from about 0 to 24 hours doesnot exceed about 0.5% (area percent bendamustine). An aspect of thisembodiment is the lyophilized powder produced from thepre-lyophilization solution or dispersion.

Still another embodiment of the invention is a bendamustinepre-lyophilization solution or dispersion comprising one or more organicsolvents where the solution or dispersions include at least onestabilizing concentration of an organic solvent which reduces the levelof degradation of bendamustine so that the amount of bendamustineethylester (as shown in Formula IV) produced during lyophilization fromabout 0 to 24 hours is no more than 0.2%, preferably 0.1%, greater thanthe concentration of bendamustine ethylester as found in the drugsubstance used to make the pre-lyophilization solution. A preferredorganic solvent is tertiary butanol.

The invention also discloses methods for preparing a bendamustinelyophilized preparation that includes dissolving bendamustine in astabilizing concentration of an alcohol solvent of between about 5% toabout 100% (v/v alcohol to form a pre-lyophilization solution; andlyophilizing the pre-lyophilization solution; wherein the bendamustinelyophilized preparation made from such methods contains not more thanabout 0.5% to about 0.9%, preferably 0.5%, (area percent ofbendamustine) HP1 as shown in Formula II, wherein said HP1 is the amountof HP1 present at release or at time zero after reconstitution of thelyophilized pharmaceutical composition of bendamustine. Other alcoholconcentrations include about 5% to about 99.9%, about 5% to about 70%,about 5% to about 60%, about 5% to about 50%, about 5% to about 40%,about 20% to about 35%. Preferred concentrations of alcohol are fromabout 20% to about 30%. Preferred alcohols include one or more ofmethanol, ethanol, propanol, iso-propanol, butanol, andtertiary-butanol. A more preferred alcohol is tertiary-butanol. Apreferred concentration of tertiary-butanol is about 20% to about 30%,preferably about 30%. An aspect of this embodiment is the addition of anexcipient before lyophilization. A preferred excipient is mannitol.Preferred pre-lyophilized concentrations of bendamustine are from about2 mg/mL to about 50 mg/mL.

In a preferred method for preparing a bendamustine lyophilizedpreparation, lyophilizing the pre-lyophilization solution comprises i)freezing the pre-lyophilization solution to a temperature below about−40° C., preferably −50° C., to form a frozen solution; ii) holding thefrozen solution at or below −40° C., preferably −50° C., for at least 2hours; iii) ramping the frozen solution to a primary drying temperaturebetween about −40° C. and about −10° C. to form a dried solution; iv)holding for about 10 to about 70 hours; v) ramping the dried solution toa secondary drying temperature between about 25° C. and about 40° C.;and vii) holding for about 5 to about 40 hours to form a bendamustinelyophilized preparation. In a more preferred method lyophilizing thepre-lyophilization solution comprises i) freezing the pre-lyophilizationsolution to about −50° C. to form a frozen solution; ii) holding thefrozen solution at about −50° C. for at least 2 hours to about 4 hours;iii) ramping to a primary drying temperature between about −20° C. andabout −12° C. to form a dried solution; iv) holding at a primary dryingtemperature for about 10 to about 48 hours; v) ramping the driedsolution to a secondary drying temperature between about 25° C. andabout 40° C.; and vi) holding at a secondary drying temperature for atleast 5 hours up to about 20 hours. A preferred alcohol istertiary-butanol. A preferred concentration of tertiary-butanol is about20% to about 30%, preferably about 30%. An aspect of this embodiment isthe addition of an excipient before lyophilization. A preferredexcipient is mannitol. Preferred pre-lyophilized concentrations ofbendamustine are from about 2 mg/mL to about 50 mg/mL.

Another embodiment of the invention is the lyophilized powder orpreparation obtained from the methods of preparing a bendamustinelyophilized preparation disclosed herein.

The invention also involves bendamustine formulations for lyophilizationthat include an excipient and a stabilizing concentration of an organicsolvent. A preferred formulation includes bendamustine at aconcentration of about 15 mg/mL, mannitol at a concentration of about25.5 mg/mL, tertiary-butyl alcohol at a concentration of about 30% (v/v)and water. Included in this embodiment of the invention are thelyophilized preparations made from such bendamustine formulations.

Included in the inventions are methods of treating a medical conditionin a patient that involve administering a therapeutically effectiveamount of a pharmaceutical composition of the invention where thecondition is amenable to treatment with said pharmaceutical composition.Some conditions amenable to treatment with the compositions of theinvention include chronic lymphocytic leukemia (CLL), Hodgkin's disease,non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), breast cancer,small cell lung cancer, hyperproliferative disorders, and an autoimmunedisease. Preferred conditions include NHL, CLL, breast cancer, and MM.Preferred autoimmune diseases include rheumatoid arthritis, multiplesclerosis or lupus.

Included in the inventions are the use of the pharmaceuticalcompositions or pharmaceutical preparations of the invention in themanufacture of a medicament for the treatment of a medical condition, asdefined herein, in a patient that involve administering atherapeutically effective amount of a pharmaceutical composition of theinvention where the condition is amenable to treatment with saidpharmaceutical composition.

Also included in the invention are methods of treating in which thepharmaceutical compositions of the invention are in combination with oneor more anti-neoplastic agents where the antineoplastic agent is givenprior, concurrently, or subsequent to the administration of thepharmaceutical composition of the invention. Preferred antineoplasticagents are antibodies specific for CD20.

Another embodiment of the invention is a lyophilization cycle forproducing lyophilized bendamustine preparations of the invention. Apreferred lyophilization cycle includes a) freezing to about −50° C.over about 8 hours; b) holding at −50° C. for about 4 hours; c) rampingto −25° C. over about 3 hours; d) holding at −10° C. for 30 hours; e)ramping to between about 25° C. and about 40° C. or higher for about 3hours; f) holding between about 25° C. and about 40° C. for about 25hours; g) ramping to about 20° C. in 1 hour; h) unloading at about 20°C., at a pressure of 13.5 psi in a pharmaceutically acceptable containerthat is hermetically sealed; wherein the pressure is about 150 micronsthroughout primary drying and 50 microns throughout secondary drying. Anaspect of this cycle involves step (e) which is ramped to about 30-35°C. for 3 hours and then ramped to 40° C. for 5 hours. Another aspect ofthis embodiment is the lyophilized powered prepared from suchlyophilization cycles. A more preferred lyophilization cycle includes i)starting with a shelf temperature of about 5° C. for loading; ii)freezing to about −50° C. over about 8 hours; iii) holding at −50° C.for about 4 hours; iv) ramping to about −20° C. over about 3 hours; v)holding at about −20° C. for 6 hours; ramping to about −15° C. overabout 1 hour; vi) holding at −15° C. for about 20 hours; vii) ramping toabout −15° C. over about 1 hour; viii) holding at about −15° C. forabout 20 hours; ix) ramping to about −12° C. over about 0.5 hours; x)holding at about −12° C. for about 15.5 hours; xi) ramping to betweenabout 25° C. and about 40° C. or higher for about 15 hours; xii) holdingbetween about 25° C. and about 40° C. for about 10 hours; xiii) rampingto about 40° C. over about 1 hour; and xiv) holding at about 40° C. forabout 5 hours; unloading at about 5° C., at a pressure of about 13.5 psiin a pharmaceutically acceptable container that is hermetically sealed;wherein the pressure is about 150 microns throughout primary drying and50 microns throughout secondary drying. In a preferred embodiment step(xi) is ramped to about 30-35° C. for about 15 hours.

The invention also encompasses a pharmaceutical dosage form ofbendamustine containing not more than about 0.5% to about 0.9%,preferably 0.5%, HP1 (area percent of bendamustine) wherein said dosageform comprises a vial or other pharmaceutically acceptable container,wherein said HP1 is the amount of HP1 present pre-reconstitution or attime zero after reconstitution of said dosage form. Preferredconcentrations of bendamustine include about 10 to about 500mg/container, about 100 mg/container, about 5 mg to about 2 g/containerand about 170 mg/container.

The present invention also includes pre-lyophilized pharmaceuticalcompositions of bendamustine. A preferred pre-lyophilized compositionincludes bendamustine HCl about 15 mg/mL, mannitol about 25.5 mg/mL,about 30% (v/v) tertiary-butyl alcohol, and water.

These and other embodiments of the invention are described hereinbelowor are evident to persons of ordinary skill in the art based on thefollowing disclosures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the solubility of bendamustine at various temperatures fortwo different solutions of bendamustine in tertiary butanol.

FIG. 2 shows the purity results of an HPLC analysis after incubatingbendamustine in various alcohols for 24 hours at 5° C. Results arepresented as the area percent of the bendamustine peak.

FIG. 3 shows HP1 (Formula II) formation after 24 hours in variousalcohol/water co-solvents at 5° C.

FIG. 4 shows dimer (Formula III) formation after 24 hours in variousalcohol/water co-solvents at 5° C.

FIG. 5—shows a lyophilization cycle for bendamustine using a TBA/waterco-solvent.

FIG. 6 shows a chromatogram for Ribomustin® using HPLC method No. 1.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “formulate” refers to the preparation of adrug, e.g., bendamustine, in a form suitable for administration to amammalian patient, preferably a human. Thus, “formulation” can includethe addition of pharmaceutically acceptable excipients, diluents, orcarriers.

As used herein, the term “lyophilized powder” or “lyophilizedpreparation” refers to any solid material obtained by lyophilization,i.e., freeze-drying of an aqueous solution. The aqueous solution maycontain a non-aqueous solvent, i.e. a solution composed of aqueous andone or more non-aqueous solvent(s). Preferably, a lyophilizedpreparation is one in which the solid material is obtained byfreeze-drying a solution composed of aqueous and one or more non-aqueoussolvents, more preferably the non-aqueous solvent is an alcohol.

By “stable pharmaceutical composition” is meant any pharmaceuticalcomposition having sufficient stability to have utility as apharmaceutical product. Preferably, a stable pharmaceutical compositionhas sufficient stability to allow storage at a convenient temperature,preferably between −20° C. and 40° C., more preferably about 2° C. toabout 30° C., for a reasonable period of time, e.g., the shelf-life ofthe product which can be as short as one month but is typically sixmonths or longer, more preferably one year or longer even morepreferably twenty-four months or longer, and even more preferablythirty-six months or longer. The shelf-life or expiration can be thatamount of time where the active ingredient degrades to a point below 90%purity. For purposes of the present invention stable pharmaceuticalcomposition includes reference to pharmaceutical compositions withspecific ranges of impurities as described herein. Preferably, a stablepharmaceutical composition is one which has minimal degradation of theactive ingredient, e.g., it retains at least about 85% of un-degradedactive, preferably at least about 90%, and more preferably at leastabout 95%, after storage at 2-30° C. for a 2-3 year period of time.

By “stable lyophilized preparation” is meant any lyophilized preparationhaving sufficient stability, such characteristics as similarly definedherein for a stable pharmaceutical composition, to have utility as apharmaceutical product

By “degraded” is meant that the active has undergone a change inchemical structure.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered that will relieve to someextent one or more of the symptoms of the disorder being treated. Inreference to the treatment of neoplasms, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor, (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis, (3) inhibiting to some extent(that is, slowing to some extent, preferably stopping) tumor growth,and/or, (4) relieving to some extent (or, preferably, eliminating) oneor more symptoms associated with the cancer. Therapeutically effectiveamount can also mean preventing the disease from occurring in an animalthat may be predisposed to the disease but does not yet experience orexhibit symptoms of the disease (prophylactic treatment). Further,therapeutically effective amount can be that amount that increases thelife expectancy of a patient afflicted with a terminal disorder. Typicaltherapeutically effective doses for bendamustine for the treatment ofnon-Hodgkin's lymphoma can be from about 60-120 mg/m² given as a singledose on two consecutive days. The cycle can be repeated about everythree to four weeks. For the treatment of chronic lymphocytic leukemia(CLL) bendamustine can be given at about 80-100 mg/m² on days 1 and 2.The cycle can be repeated after about 4 weeks. For the treatment ofHodgkin's disease (stages II-IV), bendamustine can be given in the“DBVBe regimen” with daunorubicin 25 mg/m² on days 1 and 15, bleomycin10 mg/m² on days 1 and 15, vincristine 1.4 mg/m² on days 1 and 15, andbendamustine 50 mg/m² on days 1-5 with repetition of the cycle aboutevery 4 weeks. For breast cancer, bendamustine (120 mg/m²) on days 1 and8 can be given in combination with methotrexate 40 mg/m² on days 1 and8, and 5-fluorouracil 600 mg/m² on days 1 and 8 with repetition of thecycle about every 4 weeks. As a second-line of therapy for breastcancer, bendamustine can be given at about 100-150 mg/m² on days 1 and 2with repetition of the cycle about every 4 weeks.

As used herein “neoplastic” refers to a neoplasm, which is an abnormalgrowth, such growth occurring because of a proliferation of cells notsubject to the usual limitations of growth. As used herein,“anti-neoplastic agent” is any compound, composition, admixture,co-mixture, or blend which inhibits, eliminates, retards, or reversesthe neoplastic phenotype of a cell.

As used herein “hyperproliferation” is the overproduction of cells inresponse to a particular growth factor. “Hyperproliferative disorders”are diseases in which the cells overproduce in response to a particulargrowth factor. Examples of such “hyperproliferative disorders” includediabetic retinopathy, psoriasis, endometriosis, cancer, maculardegenerative disorders and benign growth disorders such as prostateenlargement.

As used herein, the term “vial” refers to any walled container, whetherrigid or flexible.

“Controlling” as used herein means putting process controls in place tofacilitate achievement of the thing being controlled. For example, in agiven case, “controlling” can mean testing samples of each lot or anumber of lots regularly or randomly; setting the concentration ofdegradants as a release specification; selecting process conditions,e.g., use of alcohols and/or other organic solvents in thepre-lyophilization solution or dispersion, so as to assure that theconcentration of degradants of the active ingredient is not unacceptablyhigh; etc. Controlling for degradants by setting release specificationsfor the amount of degradants can be used to facilitate regulatoryapproval of a pharmaceutical product by a regulatory agency, such as theU.S. Food and Drug Administration and similar agencies in othercountries or regions (“agency”).

The term “pharmaceutically acceptable” as used herein means that thething that is pharmaceutically acceptable, e.g., components, includingcontainers, of a pharmaceutical composition, does not cause unacceptableloss of pharmacological activity or unacceptable adverse side effects.Examples of pharmaceutically acceptable components are provided in TheUnited States Pharmacopeia (USP), The National Formulary (NF), adoptedat the United States Pharmacopeial Convention, held in Rockville, Md. in1990 and FDA Inactive Ingredient Guide 1990, 1996 issued by the U.S.Food and Drug Administration (both are hereby incorporated by referenceherein, including any drawings). Other grades of solutions or componentsthat meet necessary limits and/or specifications that are outside of theUSP/NF may also be used.

The term “pharmaceutical composition” as used herein shall mean acomposition that is made under conditions such that it is suitable foradministration to humans, e.g., it is made under GMP conditions andcontains pharmaceutically acceptable excipients, e.g., withoutlimitation, stabilizers, bulking agents, buffers, carriers, diluents,vehicles, solubilizers, and binders. As used herein pharmaceuticalcomposition includes but is not limited to a pre-lyophilization solutionor dispersion as well as a liquid form ready for injection or infusionafter reconstitution of a lyophilized preparation.

A “pharmaceutical dosage form” as used herein means the pharmaceuticalcompositions disclosed herein being in a container and in an amountsuitable for reconstitution and administration of one or more doses,typically about 1-2, 1-3, 1-4, 1-5, 1-6, 1-10, or about 1-20 doses.Preferably, a “pharmaceutical dosage form” as used herein means alyophilized pharmaceutical composition disclosed herein in a containerand in an amount suitable for reconstitution and delivery of one or moredoses, typically about 1-2, 1-3, 1-4, 1-5, 1-6, 1-10, or about 1-20doses. The pharmaceutical dosage form can comprise a vial or syringe orother suitable pharmaceutically acceptable container. The pharmaceuticaldosage form suitable for injection or infusion use can include sterileaqueous solutions or dispersions or sterile powders comprising an activeingredient which are adapted for the extemporaneous preparation ofsterile injectable or infusible solutions or dispersions. In all cases,the ultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol such as glycerol, propylene glycol, or liquidpolyethylene glycols and the like, vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The prevention of the growth ofmicroorganisms can be accomplished by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

As used herein, the term “excipient” means the substances used toformulate active pharmaceutical ingredients (API) into pharmaceuticalformulations; in a preferred embodiment, an excipient does not lower orinterfere with the primary therapeutic effect of the API. Preferably, anexcipient is therapeutically inert. The term “excipient” encompassescarriers, diluents, vehicles, solubilizers, stabilizers, bulking agents,and binders. Excipients can also be those substances present in apharmaceutical formulation as an indirect or unintended result of themanufacturing process. Preferably, excipients are approved for orconsidered to be safe for human and animal administration, i.e., GRASsubstances (generally regarded as safe). GRAS substances are listed bythe Food and Drug administration in the Code of Federal Regulations(CFR) at 21 CFR §182 and 21 CFR §184, incorporated herein by reference.Preferred excipients include, but are not limited to, hexitols,including mannitol and the like.

As used herein “a stabilizing concentration of an organic solvent” or “astabilizing concentration of an alcohol” means that amount of an organicsolvent or alcohol that reduces the level of degradation of bendamustineto achieve a specified level of degradants in the final drug product.For example, with respect to the degradant HP1, a stabilizingconcentration of an organic solvent is that amount which results in anHP1 concentration (area percent of bendamustine) of less than about0.5%, preferably less than 0.45%, preferably less than 0.40%, morepreferably less than 0.35%, more preferably less than 0.30%, and evenmore preferably less than 0.25%. With respect to the overall or totaldegradant concentration of the final drug product, a stabilizingconcentration of an organic solvent is that amount that results in atotal degradant concentration (at the time of drug product release) ofless than about 7% (area percent bendamustine), preferably less thanabout 6%, more preferably less than about 5%, and even more preferablyless than about 4.0%. By “area percent of bendamustine” is meant theamount of a specified degradant, e.g., HP1, relative to the amount ofbendamustine as determined, e.g., by HPLC.

The term “organic solvent” means an organic material, usually a liquid,capable of dissolving other substances.

As used herein, “trace amount of an organic solvent” means an amount ofsolvent that is equal to or below recommended levels for pharmaceuticalproducts, for example, as recommended by ICH guidelines (InternationalConferences on Harmonization, Impurities—Guidelines for ResidualSolvents. Q3C. Federal Register. 1997; 62(247):67377). The lower limitis the lowest amount that can be detected.

The term “release” or “at release” means the drug product has met therelease specifications and can be used for its intended pharmaceuticalpurpose.

A. General

The invention provides stable, pharmaceutically acceptable compositionsprepared from bendamustine. In particular, the invention providesformulations for the lyophilization of bendamustine HCl. The lyophilizedpowder obtained from such formulations is more easily reconstituted thanthe presently available lyophilized powder of bendamustine. Further, thelyophilized products of the present invention have a better impurityprofile than Ribomustin® with respect to certain impurities, inparticular HP1, bendamustine dimer, and bendamustine ethylester, priorto reconstitution, upon storage of the lyophilate, or followingreconstitution and admixture.

The present invention further provides formulations of bendamustineuseful for treating neoplastic diseases. The formulations describedherein can be administered alone or in combination with at least oneadditional anti-neoplastic agent and/or radioactive therapy.

An aspect of the invention is conditions and means for enhancing thestability of bendamustine prior to and during the lyophilizationprocess, upon shelf storage or upon reconstitution.

Anti-neoplastic agents which may be utilized in combination with theformulations of the invention include those provided in the Merck Index11, pp 16-17, Merck & Co., Inc. (1989) and The Chemotherapy Source Book(1997). Both books are widely recognized and readily available to theskilled artisan.

There are large numbers of antineoplastic agents available in commercialuse, in clinical evaluation and in pre-clinical development, which couldbe selected for treatment of neoplasia by combination drug chemotherapy.Such antineoplastic agents fall into several major categories, namely,antibiotic-type agents, covalent DNA-binding drugs, antimetaboliteagents, hormonal agents, including glucocorticoids such as prednisoneand dexamethasone, immunological agents, interferon-type agents,differentiating agents such as the retinoids, pro-apoptotic agents, anda category of miscellaneous agents, including compounds such asantisense, small interfering RNA, and the like. Alternatively, otheranti-neoplastic agents, such as metallomatrix proteases (MMP)inhibitors, SOD mimics or alpha_(v) beta₃ inhibitors may be used.

One family of antineoplastic agents which may be used in combinationwith the compounds of the inventions consists of antimetabolite-typeantineoplastic agents. Suitable antimetabolite antineoplastic agents maybe selected from the group consisting of alanosine, AG2037 (Pfizer),5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium,carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabinephosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC,dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC,doxifluridine, Wellcome EHNA, Merck & Co. EX-015, fazarabine,floxuridine, fludarabine phosphate, 5-fluorouracil,N-(2′-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, isopropylpyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim,methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCINSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA,pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, TakedaTAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosinekinase inhibitors, tyrosine protein kinase inhibitors, Taiho UFT anduricytin.

A second family of antineoplastic agents which may be used incombination with the compounds of the invention consists of covalentDNA-binding agents. Suitable alkylating-type antineoplastic agents maybe selected from the group consisting of Shionogi 254-S,aldo-phosphamide analogues, altretamine, anaxirone, Boehringer MannheimBBR-2207, bestrabucil, budotitane, Wakunaga CA-102, carboplatin,carmustine, Chinoin-139, Chinoin-153, chlorambucil, cisplatin,cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233, cyplatate,Degussa D-19-384, Sumimoto DACHP(Myr)2, diphenylspiromustine, diplatinumcytostatic, Erba distamycin derivatives, Chugai DWA-2114R, ITI E09,elmustine, Erbamont FCE-24517, estramustine phosphate sodium,fotemustine, Unimed G-6-M, Chinoin GYKI-17230, hepsul-fam, ifosfamide,iproplatin, lomustine, mafosfamide, melphalan, mitolactol, Nippon KayakuNK-121, NCI NSC-264395, NCI NSC-342215, oxaliplatin, Upjohn PCNU,prednimustine, Proter PTT-119, ranimustine, semustine, SmithKlineSK&F-101772, Yakult Honsha SN-22, spiromustine, Tanabe Seiyaku TA-077,tauromustine, temozolomide, teroxirone, tetraplatin and trimelamol.

Another family of antineoplastic agents which may be used in combinationwith the compounds disclosed herein consists of antibiotic-typeantineoplastic agents. Suitable antibiotic-type antineoplastic agentsmay be selected from the group consisting of Taiho 4181-A, aclarubicin,actinomycin D, actinoplanone, alanosine, Erbamont ADR-456, aeroplysininderivative, Ajinomoto AN-201-II, Ajinomoto AN-3, Nippon Sodaanisomycins, anthracycline, azino-mycin-A, bisucaberin, Bristol-MyersBL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-MyersBMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, bleomycinsulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoximycin,dactinomycin, daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79, KyowaHakko DC-88A, Kyowa Hakko DC89-A1, Kyowa Hakko DC92-B, ditrisarubicin B,Shionogi DOB-41, doxorubicin, doxorubicin-fibrinogen, elsamicin-A,epirubicin, erbstatin, esorubicin, esperamicin-A1, esperamicin-A1b,Erbamont FCE-21954, Fujisawa FK-973, fostriecin, Fujisawa FR-900482,glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins,kazusamycin, kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602,Kyowa Hakko KT-5432, Kyowa Hakko KT-5594, Kyowa Hakko KT-6149, AmericanCyanamid LL-D49194, Meiji Seika ME 2303, menogaril, mitomycin,mitoxantrone, SmithKline M-TAG, neoenactin, Nippon Kayaku NK-313, NipponKayaku NKT-01, SRI International NSC-357704, oxalysine, oxaunomycin,peplomycin, pilatin, pirarubicin, porothramycin, pyrindamycin A, TobishiRA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin,Sumitomo SM-5887, Snow Brand SN-706, Snow Brand SN-07, sorangicin-A,sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SSPharmaceutical SS-9816B, steffimycin B, Taiho 4181-2, talisomycin,Takeda TAN-868A, terpentecin, thrazine, tricrozarin A, Upjohn U-73975,Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024 andzorubicin.

A fourth family of antineoplastic agents which may be used incombination with the compounds of the invention include a miscellaneousfamily of antineoplastic agents selected from the group consisting ofalpha-carotene, alpha-difluoromethyl-arginine, acitretin, arsenictrioxide, Avastin® (bevacizumab), Biotec AD-5, Kyorin AHC-52, alstonine,amonafide, amphethinile, amsacrine, Angiostat, ankinomycin,anti-neoplaston A10, antineoplaston A2, antineoplaston A3,antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolinglycinate, asparaginase, Avarol, baccharin, batracylin, benfluoron,benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-MyersBMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-502, WellcomeBW-773, caracemide, carmethizole hydrochloride, Ajinomoto CDAF,chlorsulfaquinoxalone, Chemes CHX-2053, Chemex CHX-100, Warner-LambertCI-921, Warner-Lambert CI-937, Warner-Lambert CI-941, Warner-LambertCI-958, clanfenur, claviridenone, ICN compound 1259, ICN compound 4711,Contracan, Yakult Honsha CPT-II, crisnatol, curaderm, cytochalasin B,cytarabine, cytocytin, Merz D-609, DABIS maleate, dacarbazine,datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone,dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, DaiichiSeiyaku DN-9693, elliprabin, elliptinium acetate, epothionesTsumuraEPMTC, erbitux, ergotamine, erlotnib, etoposide, etretinate,fenretinide, Fujisawa FR-57704, gallium nitrate, genkwadaphnin, Gleevec®(imatnib), Chugai GLA-43, Glaxo GR-63178, gefitinib, grifolan NMF-5N,hexadecylphosphocholine, Green Cross HO-221, homoharringtonine,hydroxyurea, BTG ICRF-187, indanocine, ilmofosine, isoglutamine,isotretinoin, Otsuka JI-36, Ramot K-477, Otsuak K-76COONa, KurehaChemical K-AM, MECT Corp KI-8110, American Cyanamid L-623, leukoregulin,lonidamine, Lundbeck LU-23-112, Lilly LY-186641, NCI (US) MAP, marycin,mefloquine, Merrel Dow MDL-27048, Medco MEDR-340, merbarone, merocyaninederivatives, methylanilinoacridine, Molecular Genetics MGI-136,minactivin, mitonafide, mitoquidone, mopidamol, motretinide, ZenyakuKogyo MST-16, N-(retinoyl)amino acids, Nisshin Flour Milling N-021,N-acylated-dehydroalanines, nafazatrom, Taisho NCU-190, nocodazolederivative, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782,NCI NSC-95580, octreotide, Ono ONO-112, oquizanocine, Akzo Org-10172,paclitaxel, pancratistatin, pazelliptine, Warner-Lambert PD-111707,Warner-Lambert PD-115934, Warner-Lambert PD-131141, Pierre FabrePE-1001, ICRT peptide D, piroxantrone, polyhaematoporphyrin, polypreicacid, Efamol porphyrin, probimane, procarbazine, proglumide, Invitronprotease nexin I, Tobishi RA-700, razoxane, Sapporo Breweries RBS,restrictin-P, retelliptine, retinoic acid, Rhone-Poulenc RP-49532,Rhone-Poulenc RP-56976, Rituxan® (and other anti CD20 antibodies, e.g.Bexxar®, Zevalin®), SmithKline SK&F-104864, statins (Lipitor® etc.),Sumitomo SM-108, Kuraray SMANCS, SeaPharm SP-10094, spatol,spirocyclopropane derivatives, spirogermanium, Unimed, SS PharmaceuticalSS-554, strypoldinone, Stypoldione, Suntory SUN 0237, Suntory SUN 2071,superoxide dismutase, Thalidomide, Thalidomide analogs, Toyama T-506,Toyama T-680, taxol, Teijin TEI-0303, teniposide, thaliblastine, EastmanKodak TJB-29, tocotrienol, Topostin, Teijin TT-82, Kyowa Hakko UCN-01,Kyowa Hakko UCN-1028, ukrain, Eastman Kodak USB-006, vinblastinesulfate, vincristine, vindesine, vinestramide, vinorelbine, vintriptol,vinzolidine, withanolides and Yamanouchi YM-534, Zometa®.

Examples of radioprotective agents which may be used in the combinationchemotherapy of this invention are AD-5, adchnon, amifostine analogues,detox, dimesna, 1-102, MM-159, N-acylated-dehydroalanines,TGF-Genentech, tiprotimod, amifostine, WR-151327, FUT-187, ketoprofentransdermal, nabumetone, superoxide dismutase (Chiron and Enzon).

Methods for preparation of the antineoplastic agents described above maybe found in the literature. Methods for preparation of doxorubicin, forexample, are described in U.S. Pat. Nos. 3,590,028 and 4,012,448.Methods for preparing metallomatrix protease inhibitors are described inEP 780386. Methods for preparing .alpha_(v) .beta₃ inhibitors aredescribed in WO 97/08174.

Preferred anti-neoplastic agents include, without limitation, one ormore of daunorubicin, bleomycin, vincristine, doxorubicin, dacarbazine,prednisolone, mitoxantrone, prednisone, methotrexate, 5-fluorouracil,dexamethasone, thalidomide, thalidomide derivatives, 2ME2, Neovastat, R11 5777, arsenic trioxide, bortezomib, tamoxifen, G3139 (antisense), andSU5416, mitomycin, anti-CD20 antibodies, such as Rituxan® andR-etodolac.

Preferred drug regimens for which the present formulation may be used inconjunction with or as a replacement for one or more of the componentsincludes, without limitation, ABVD (doxorubicin, bleomycin, vincristine,dacarbazine), DBV (daunorubicin, belomycin, vincristine), CVPP(cyclophosphamide, vinblastine, procarbazine, prednisolone), COP(cyclophosphamide, vincristine, prednisolone), CHOP (cyclophosphamide,doxorubicin,

vincristine and prednisone) and CMF (cyclophosphamide, methotrexate,5-fluorouracil). Additional regimens are given in Table A below.

TABLE A Cancer Therapeutic Regimens Abbreviation Drugs Used Disease ACDoxorubicin & Breast cancer Cyclophosphamide CFM (CF, FNC)Cyclophosphamide, Breast cancer Fluorouracil, Mitaxantrone CMFCyclophosphamide, Breast cancer Methotrexate, Fluorouracil NFLMitoxantrone, Fluorouracil, Breast cancer Leucovorin SequentialDoxorubicin Breast cancer Dox-CMF VATH Vinblastine, Doxorubicin, Breastcancer Thiotepa, Fluoxymesterone EMA-86 Etoposide, Mitoxantrone, AML(induction) Ctyarabine 7 + 3 Cytarabine WITH AML (induction)Daunorubicin OR Idarobicin OR Mitoxantrone 5 + 2 Cytarabine WITH AML(induction) Daunorubicin OR Mitoxantrone HiDAC Cytarabine AML(post-remission) ABVD Doxorubicin, Bleomycin, Hodgkin's Vinblastine,Dacarbazine ChlVPP Chlorambucil, Vinblastine, Hodgkin's Procarbazine,Prednisone EVA Etoposide, Vinblastine, Hodgkin's Doxorubicin MOPPMechlorethamine, Hodgkin's Vincristine, Procarbazine, PrednisoneMOPP/ABV Mechlorethamine, Hodgkin's Hybrid Vincristine, Procarbazine,Prednisone, Doxorubicin, Bleomycin, Vinblastine MOPP/ABVDMechlorethamine, Hodgkin's Doxorubicin, Vinblastine, Bleomycin,Etoposide, Prednisone CNOP Cyclophosphamide, Non-Hodgkin's Mitoxantrone,Vincristine, Prednisone COMLA Cyclophosphamide, Non-Hodgkin'sVincristine, Methotrexate, Leucovorin, Cytarabine DHAP Dexamethasone,Cisplatin, Non-Hodgkin's Cytarabine ESHAP Etoposide, Non-Hodgkin'sMethylprednisilone, Cisplatin, Cytarabine MACOP-B Methotrexate,Leucovorin, Non-Hodgkin's Doxorubicin, Cyclophosphamide, Vincristine,Prednisone, Bleomycin, Septra, Ketoconazole m-BACOD Methotrexate,Leucovorin, Non-Hodgkin's Bleomycin, Doxorubicin, Cyclophosphamide,Vincristine, Dexamethasone MINE-ESHAP Mesna, Ifosfamide, Non-Hodgkin'sMitoxantrone, Etoposide NOVP Mitoxantrone, Vinblastine, Non-Hodgkin'sPrednisone, Vincristine ProMACE/ Prednisone, Doxorubicin, Non-Hodgkin'scytaBOM Cyclophosphamide, Etoposide, Cytarabine, Bleomycin, Vincristine,Methotrexate, Leucovorin, Septra M2 Vincristine, Carmustine, MultipleMyeloma Cyclophosphamide, Melphalan, Prednisone MP Melphalan, PrednisoneMultiple Myeloma VAD Vincristine, Doxorubicin, Multiple MyelomaDexamethasone VBMCP Vincristine, Carmustine, Multiple Myeloma Melphalan,Cyclophosphamide, Prednisone

As described herein, a lyophilized formulation of bendamustine isachieved following removal of an organic solvent in water. The mosttypical example of the solvent used to prepare this formulation istertiary butanol (TBA). Other organic solvents can be used includingethanol, n-propanol, n-butanol, isoproponal, ethyl acetate, dimethylcarbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methylisobutyl ketone, acetone, 1-pentanol, methyl acetate, methanol, carbontetrachloride, dimethyl sulfoxide, hexafluoroacetone, chlorobutanol,dimethyl sulfone, acetic acid, cyclohexane. These preceding solvents maybe used individually or in combination. Useful solvents must form stablesolutions with bendamustine and must not appreciably degrade ordeactivate the API. The solubility of bendamustine in the selectedsolvent must be high enough to form commercially useful concentrationsof the drug in solvent. Additionally, the solvent should be capable ofbeing removed easily from an aqueous dispersion or solution of the drugproduct, e.g., through lyophilization or vacuum drying. Preferably, asolution having a concentration of about 2-80 mg/mL, preferably about 5to 40 mg/mL, more preferably 5-20 mg/mL and even more preferably 12 to17 mg/mL bendamustine is used.

A pharmaceutically acceptable lyophilization excipient can be dissolvedin the aqueous phase. Examples of excipients useful for the presentinvention include, without limitation, sodium or potassium phosphate,citric acid, tartaric acid, gelatin, glycine, and carbohydrates such aslactose, sucrose, maltose, glycerin, dextrose, dextran, trehalose andhetastarch. Mannitol is a preferred excipient. Other excipients that maybe used if desired include antioxidants, such as, without limitation,ascorbic acid, acetylcysteine, cysteine, sodium hydrogen sulfite,butyl-hydroxyanisole, butyl-hydroxytoluene or alpha-tocopherol acetate,or chelators.

A typical formulation and lyophilization cycle useful in accordance withthe present invention is provided below. Lyophilization can be carriedout using standard equipment as used for lyophilization or vacuumdrying. The cycle may be varied depending upon the equipment andfacilities used for the fill/finish.

In accordance with a typical embodiment of the present invention, anaqueous pre-lyophilization solution or dispersion is first formulated ina pharmaceutically acceptable compounding vessel. The solution isaseptically filtered into a sterile container, filled into anappropriate sized vial, partially stoppered and loaded into thelyophilizer. Using lyophilization techniques described herein thesolution is lyophilized until a moisture content in the range of about0.1 to about 8.0 percent is achieved. The resulting lyophilizationpowder is stable as a lyophilized powder for about six months to greaterthan about 2 years, preferably greater than about 3 years at about 5° C.to about 25° C. and can be readily reconstituted with Sterile Water forInjection, or other suitable carrier, to provide liquid formulations ofbendamustine, suitable for internal administration e.g., by parenteralinjection. For intravenous administration, the reconstituted liquidformulation, i.e., the pharmaceutical composition, is preferably asolution.

The pre-lyophilization solution or dispersion normally is firstformulated in a pharmaceutically acceptable container by: 1) adding anexcipient, such as mannitol (about 0 to about 50 mg/mL) with mixing towater (about 65% of the total volume) at ambient temperature, 2) addingan organic solvent (0.5-99.9% v/v), such as TBA to the aqueous solutionwith mixing at about 20°-35° C., 4) adding bendamustine HCl to thedesired concentration with mixing, 5) adding water to achieve the finalvolume, and 6) cooling the solution to about 1° C. to about 30° C.,preferably about 5° C. Although the preceding steps are shown in acertain order, it is understood that one skilled in the art can changethe order of the steps and quantities as needed. Quantities can beprepared on a weight basis also.

The pre-lyophilization solution or dispersion can be sterilized prior tolyophilization, sterilization is generally performed by asepticfiltration, e.g., through a 0.22 micron or less filter. Multiplesterilization filters can be used. Sterilization of the solution ordispersion can be achieved by other methods known in the art, e.g.,radiation.

In this case, after sterilization, the solution or dispersion is readyfor lyophilization. Generally, the filtered solution will be introducedinto a sterile receiving vessel, and then transferred to any suitablecontainer or containers in which the formulation may be effectivelylyophilized. Usually the formulation is effectively and efficientlylyophilized in the containers in which the product is to be marketed,such as, without limitation, a vial, as described herein and as known inthe art.

A typical procedure for use in lyophilizing the pre-lyophilizationsolutions or dispersions is set forth below. However, a person skilledin the art would understand that modifications to the procedure orprocess may be made depending on such things as, but not limited to, thepre-lyophilization solution or dispersion and lyophilization equipment.

Initially, the product is placed in a lyophilization chamber under arange of temperatures and then subjected to temperatures well below theproduct's freezing point, generally for several hours. Preferably, thetemperature will be at or below about −40° C. for at least 2 hours.After freezing is complete, the chamber and the condenser are evacuatedthrough vacuum pumps, the condenser surface having been previouslychilled by circulating refrigerant. Preferably, the condenser will havebeen chilled below the freezing point of the solution preferably toabout −40°, more preferably to about −50° C. or lower, even morepreferably to about −60° C. or lower. Additionally, evacuation of thechamber should continue until a pressure of about 10 to about 600microns, preferably about 50 to about 150 microns is obtained.

The product composition is then warmed under vacuum in the chamber andcondenser. This usually will be carried out by warming the shelveswithin the lyophilizer on which the product rests during thelyophilization process at a pressure ranging from about 10 to about 600microns. The warming process will optimally take place very gradually,over the course of several hours. For example, the product temperatureshould initially be increased from about −30° C. to about −10° C. andmaintained for about 10-70 hours. Additionally, the product temperaturecan be increased from the freezing temperature to about 25° C.-40° C.over a period of 30-192 hours. To prevent powder ejection of thelyophilate from vials, complete removal of the organic solvent and watershould be done during the initial drying phase. Complete drying can beconfirmed by stabilization of vacuum, condenser temperature and productshelf temperature. After the initial drying, the product temperatureshould be increased to about 25° C.-40° C. and maintained for about 5-40hours.

Once the drying cycle is completed, the pressure in the chamber can beslowly released to atmospheric pressure (or slightly below) withsterile, dry-nitrogen gas (or equivalent gas). If the productcomposition has been lyophilized in containers such as vials, the vialscan be stoppered, removed and sealed. Several representative samples canbe removed for purposes of performing various physical, chemical, andmicrobiological tests to analyze the quality of the product.

The lyophilized bendamustine formulation is typically marketed inpharmaceutical dosage form. The pharmaceutical dosage form of thepresent invention, although typically in the form of a vial, may be anysuitable container, such as ampoules, syringes, co-vials, which arecapable of maintaining a sterile environment. Such containers can beglass or plastic, provided that the material does not interact with thebendamustine formulation. The closure is typically a stopper, mosttypically a sterile rubber stopper, preferably a bromobutyl rubberstopper, which affords a hermetic seal.

After lyophilization, the bendamustine lyophilization powder may befilled into containers, such as vials, or alternatively thepre-lyophilization solution can be filled into such vials andlyophilized therein, resulting in vials which directly contain thelyophilized bendamustine formulation. Such vials are, after filling orlyophilization of the solution therein, sealed, as with a stopper, toprovide a sealed, sterile, pharmaceutical dosage form. Typically, a vialwill contain a lyophilized powder including about 10-500 mg/vial,preferably about 100 mg/vial, bendamustine and about 5 mg-2 g/vial,preferably about 170 mg/vial, mannitol.

The lyophilized formulations of the present invention may bereconstituted with water, preferably Sterile Water for Injection, orother sterile fluid such as co-solvents, to provide an appropriatesolution of bendamustine for administration, as through parenteralinjection following further dilution into an appropriate intravenousadmixture container, for example, normal saline.

B. Solubility

The solubility of bendamustine HCl (bendamustine) in water (alone) andwith varying amounts of alcohols commonly used in lyophilization, e.g.,methanol, ethanol, propanol, isopropanol, butanol and tertiary-butylalcohol (TBA) was determined by visual inspection. Amounts ofbendamustine at 15 mg/mL, combined with mannitol at 25.5 mg/mL wereprepared in 10 mL of the indicated alcohol solutions at room temperature(see Table 1). Samples were then refrigerated at 5° C. and inspectedafter 0, 3, 6 and 24 hours for particulates and/or precipitates.

The results shown in Table 1 indicate that bendamustine solubility isdependant on temperature and the amount of alcohol in aqueous solutions.For the alcohols tested, the solubility of bendamustine increased as theconcentration of alcohol increased. The formation of a precipitant wasalso dependent on the temperature and time. Bendamustine did notprecipitate immediately with any alcohol, but crystallized after storageat 5° C. Alcohols varied in their effect on solubility. Without wishingto be bound to any particular theory, smaller alcohols such as methanoland ethanol have less of an effect on solubility as compared with largeralcohols (tertiary-butanol and n-butanol). However, the shape of thealcohol is also important. For example n-propanol was found to be betterthan iso-propanol in preventing precipitation in this system. The twoalcohols with the greatest effect on solubility were n-propanol andtertiary-butanol.

TABLE 1 Bendamustine solubility over a 24 hour period in variousalcohols when stored at 5° C. Zero Time 3 Hours 6 Hours 24 HoursMethanol (v/v) 0% (Water Only) CCS CCS Precipitate Precipitate  5% CCSCCS Precipitate Precipitate 10% CCS CCS CCS Precipitate 20% CCS CCS CCSPrecipitate 30% CCS CCS CCS CCS Ethanol (v/v) 1.9%  CCS CCS PrecipitatePrecipitate  5% CCS CCS Precipitate Precipitate 10% CCS CCS CCSPrecipitate 20% CCS CCS CCS CCS 30% CCS CCS CCS CCS n-Propanol (v/v/) 5% CCS CCS CCS Precipitate 10% CCS CCS CCS CCS 20% CCS CCS CCS CCS 30%CCS CCS CCS CCS Iso-propanol (v/v)  5% CCS Precipitate PrecipitatePrecipitate 10% CCS CCS CCS CCS 20% CCS CCS CCS CCS 30% CCS CCS CCS CCSn-Butanol (v/v)  5% CCS CCS CCS CCS 10% CCS CCS CCS CCS 20% 2 layers 2layers 2 layers 2 layers 30% 2 layers 2 layers 2 layers 2 layersTert-Butanol (v/v)  5% CCS CCS CCS Precipitate 10% CCS CCS CCSPrecipitate 20% CCS CCS CCS CCS 30% CCS CCS CCS CCS CCS stands for clearcolorless solution

Experiments to quantitatively determine the solubility of bendamustineat various temperatures for three different solutions are summarized inFIG. 1 and Table 2. The amount of TBA, 20% (v/v) and 30% (v/v), used inthe experiment was based on stability studies (results described below).For both solutions tested, the solubility of bendamustine decreasedlinearly with temperatures from 25° C. to 0° C. This experimentconfirmed the data shown in Table 1 and highlights the difference inbendamustine solubility for 20% and 30% TBA solutions.

TABLE 2 Solubility of bendamustine in TBA −8° C. 0° C. 5° C. 25° C. 20%(v/v) TBA 25.5 mg/mL mannitol 14 mg/mL 11 mg/mL 17 mg/mL 47 mg/mL Water,q.s. to desired volume 30% (v/v) TBA 25.5 mg/mL mannitol 20 mg/mL 18mg/mL 27 mg/mL 65 mg/mL Water, q.s. to desired volume

C. Stability

Because of its instability in aqueous solutions due to hydrolysis withwater, bendamustine requires lyophilization in order to make a productsuitable for pharmaceutical use. However, during the manufacturing oflyophilized drug products, aqueous solutions are commonly needed forfilling, prior to lyophilization. Thus, the use of aqueous solutionsduring the compounding and fill processes for bendamustine and othernitrogen mustards can result in degradation of the drug product.Consequently, the effect of various alcohols on the degradation ofbendamustine was evaluated to determine if formulations could be foundthat would allow longer fill-finish times, provide lyophilate powdersthat could be reconstituted more quickly than the current Ribomustin®formulation, and/or provide lyophilized preparations of bendamustinewith a better impurity profile with respect to certain impurities, e.g.,HP1, and BM1 dimer than Ribomustin®.

Preferably, a lyophilized preparation of the invention is stable withrespect to HP1, i.e., the amount of HP1 does not increase appreciably(does not exceed the shelf-life specifications), for 6 months, morepreferably 12 months, and even more preferably greater than 24 months,e.g., 36 months, when stored at about 2° C. to about 30° C., preferably5° C.

Table 3 shows the stability results of bendamustine in water with noaddition of alcohol over a 24 hour period at 5° C. Bendamustine degradesrapidly in water alone and forms predominantly the hydrolysis product,HP1 (monohydroxy bendamustine).

TABLE 3 Stability of bendamustine in water Hold Time Purity (% Area) HP1(%) Dimer (%) 0% Alcohol, i.e., 0 hours 99.11 0.60 0.11 Water Alone 3hours 98.83 0.86 0.13 6 hours 98.44 1.22 0.17 24 hours  95.67 3.81 0.29

The other major degradant observed during this study and other long termstability studies was the dimer of bendamustine.

Other degradants contained in the Ribomustin lyophilized product arebendamustine ethylester (BM1EE) (Formula IV) and BM1DCE (Formula V).BM1EE is formed when bendamustine reacts with ethyl alcohol.

FIG. 2 summarizes the purity results of an HPLC analysis afterincubating bendamustine in various alcohols for 24 hours at 5° C.Results are presented as the area percent of the total peak area. Thenumerical values for FIG. 2 are provided in Tables 3-9. The purity washighest in solutions containing higher concentration of alcohols,regardless of the alcohol. Of the alcohols evaluated, bendamustinedegraded the least in a solution containing about 30% (v/v) TBA. Inabout 10% and about 20% alcohol solutions, n-butanol was superior inpreventing degradation of bendamustine. At 20% and 30% (v/v), n-butanolin water resulted in a biphasic system due to the insolubility ofn-butanol in water at these concentrations.

FIGS. 3 and 4 show the amount of degradation of bendamustine as measuredby HP1 and dimer formation quantified by HPLC (as described herein). HP1and dimer formation increased as the amount of alcohol concentrationdecreased regardless of the alcohol. This increase in impuritiesoccurred with an anticipated time dependence (see Tables 3-9).Tert-butanol and n-butanol appeared superior to other alcohols inpreventing degradation of the product. As seen in Table 10, mannitol hadno effect on the stabilization of bendamustine with TBA.

TABLE 4 HPLC stability results for the stability of bendamustine invarious ethyl alcohol concentrations over a 24 hour period. HP1 andDimer were impurities that increased in this study. V/V alcohol HoldTime Purity (% Area) HP1 (%) Dimer (%) 1.9% Ethanol 0 hours 99.11 0.640.12 3 hours 98.83 0.90 0.14 6 hours 98.60 1.12 0.15 24 hours  96.163.41 0.27 5% Ethanol 0 hours 99.31 0.44 0.12 3 hours 99.10 0.64 0.13 6hours 98.87 0.86 0.14 24 hours  96.89 2.68 0.25 10% Ethanol 0 hours99.44 0.33 0.11 3 hours 99.28 0.48 0.12 6 hours 99.10 0.65 0.12 24hours  98.03 1.57 0.18 20% Ethanol 0 hours 99.54 0.22 0.10 3 hours 99.450.30 0.11 6 hours 99.36 0.39 0.11 24 hours  98.61 0.96 0.15 30% Ethanol0 hours 99.62 0.15 0.10 3 hours 99.56 0.21 0.11 6 hours 99.52 0.24 0.1224 hours  99.21 0.45 0.12

TABLE 5 HPLC stability results for bendamustine in various Tert- butanolconcentrations over a 24 hour period. HP1 and Dimer were impurities thatincreased in this study. Concentration alcohol (v/v) Hold Time Purity (%Area) HP1 (%) Dimer (%) 5% Tert-butanol 0 hours 99.34 0.41 0.12 3 hours99.10 0.64 0.14 6 hours 98.85 0.88 0.13 24 hours  97.58 2.09 0.20 10%Tert-butanol 0 hours 99.46 0.30 0.11 3 hours 99.26 0.48 0.12 6 hours99.05 0.69 0.13 24 hours  98.04 1.64 0.19 20% Tert-butanol 0 hours 99.590.17 0.11 3 hours 99.48 0.29 0.11 6 hours 99.35 0.40 0.12 24 hours 98.35 1.27 0.20 30% Tert-butanol 0 hours 99.63 0.13 0.10 3 hours 99.600.16 0.10 6 hours 99.58 0.18 0.11 24 hours  99.42 0.34 0.12

TABLE 6 HPLC stability results for various n-propyl alcoholconcentrations over a 24 hour period. HP1 and Dimer were impurities thatincreased in this study. Concentration alcohol (v/v) Hold Time Purity (%Area) HP1 (%) Dimer (%) 5% n-Propanol 0 hours 99.25 0.43 0.13 3 hours99.00 0.66 0.15 6 hours 98.72 0.94 0.16 24 hours  97.24 2.33 0.26 10%n-Propanol 0 hours 99.34 0.33 0.15 3 hours 99.17 0.48 0.14 6 hours 98.920.70 0.16 24 hours  97.67 1.83 0.28 20% n-Propanol 0 hours 99.45 0.330.13 3 hours 99.42 0.26 0.13 6 hours 99.29 0.39 0.14 24 hours  98.600.97 0.24 30% n-Propanol 0 hours 99.53 0.15 0.13 3 hours 99.51 0.15 0.156 hours 99.44 0.20 0.11 24 hours  99.27 0.36 0.17

TABLE 7 HPLC stability results for bendamustine in various iso- propylalcohol concentrations over a 24 hour period. HP1 and Dimer wereimpurities that increased in this study. Concentration alcohol (v/v)Hold Time Purity (% Area) HP1 (%) Dimer (%) 5% Iso-propanol 0 hours99.21 0.48 0.13 3 hours 98.65 0.72 0.14 6 hours 98.56 1.02 0.14 24hours  96.14 3.35 0.26 10% Iso-propanol 0 hours 99.32 0.37 0.12 3 hours99.11 0.55 0.14 6 hours 98.85 0.75 0.16 24 hours  97.68 1.92 0.21 20%Iso-propanol 0 hours 99.49 0.21 0.11 3 hours 99.39 0.31 0.12 6 hours99.22 0.42 0.13 24 hours  98.61 1.04 0.17 30% Iso-propanol 0 hours 99.560.15 0.10 3 hours 99.47 0.20 0.12 6 hours 99.40 0.24 0.11 24 hours 99.15 0.52 0.14

TABLE 8 HPLC stability results for bendamustine in various methylalcohol concentrations over a 24 hour period. HP1 and Dimer wereimpurities that increased in this study. Concentration alcohol (v/v)Hold Time Purity (% Area) HP1 (%) Dimer (%) 5% Methanol 0 hours 99.350.40 0.12 3 hours 98.97 0.70 0.14 6 hours 98.66 0.95 0.14 24 hours 96.65 2.83 0.23 10% Methanol 0 hours 99.42 0.34 0.11 3 hours 99.01 0.590.12 6 hours 98.86 0.80 0.12 24 hours  97.65 1.85 0.18 20% Methanol 0hours 99.56 0.22 0.11 3 hours 99.31 0.38 0.11 6 hours 98.99 0.50 0.12 24hours  98.31 1.15 0.16 30% Methanol 0 hours 99.59 0.18 0.10 3 hours99.43 0.27 0.11 6 hours 99.25 0.34 0.11 24 hours  98.65 0.76 0.13

TABLE 9 HPLC stability results for bendamustine in various n-butylalcohol concentrations over a 24 hour period. HP1 and Dimer wereimpurities that increased in this study. Concentration alcohol (v/v)Hold Time Purity (% Area) HP1 (%) Dimer (%) 5% Butanol 0 hours 99.250.49 0.13 3 hours 98.94 0.73 0.14 6 hours 98.76 0.91 0.14 24 hours 97.46 2.20 0.21 10% Butanol 0 hours 99.44 0.30 0.11 3 hours 99.18 0.490.12 6 hours 99.03 0.64 0.12 24 hours  98.13 1.55 0.17 20% Butanol^(a) 0hours 99.54 0.23 0.10 3 hours 99.45 0.31 0.11 6 hours 99.30 0.40 0.11 24hours  98.81 0.91 0.14 30% Butanol^(a) 0 hours 99.55 0.24 0.10 3 hours99.40 0.29 0.10 6 hours 99.40 0.37 0.11 24 hours  99.00 0.74 0.12^(a)Both solutions had 2 layers/phases of liquids in the vial. Solutionswere vortexed prior to sample preparation.

The results in Tables 1-9 indicate that the stability of bendamustineHCl with respect to HP1 and dimer improves with increasing alcoholconcentration.

TABLE 10 HPLC stability results for bendamustine in TBA with and withoutmannitol over a 24 hour period. Sample Purity (% Area) HP1 (%) TBA 20%(v/v) with Mannitol 0 hours 99.59 0.17 24 hours @ 5° C. 99.35 1.27 TBA20% (v/v) without Mannitol 0 hours 100.0 0.00 24 hours @ 5° C. 98.801.21 NOTE: The samples analyzed without mannitol were analyzed by HPLCusing a normal phase method while the samples analyzed with mannitolused a reverse phase HPLC method. Slight variability may be seen inother samples analyzed between the two methods.

D. Lyophilization Cycle Development

Different pre-lyophilization formulations were prepared at variousconcentrations of bendamustine, mannitol, and alcohols in water. Thecycle development was changed and optimized at each step for freezing(fast vs. slow), primary drying (both temperature and pressure), andsecondary drying as described herein.

Based upon all of the information detailed above on solubility,stability, and ease of lyophilization, preferred formulations includethe following:

Ingredients Concentration Bendamustine about 2-40 mg/mL Mannitol about0-50 mg/mL Alcohol about 0.5%-40% (v/v) Water, q.s. to desired volume

wherein the alcohol is selected from methanol, n-propanol, orisopropanol

Ingredients Concentration Bendamustine about 5-20 mg/mL Mannitol 10-30mg/mL Alcohol 1-20% (v/v) Water, q.s. to desired volume

wherein the alcohol is selected from methanol, n-propanol, orisopropanol

Ingredients Concentration Bendamustine about 5-20 mg/mL Mannitol 10-30mg/mL Alcohol 5-40% (v/v) Water, q.s. to desired volume

Ingredients Concentration Bendamustine HCl about 12-17 mg/mL Mannitolabout 20-30 mg/mL Alcohol about 5-15% (v/v) Water, q.s. to desiredvolume

Ingredients Concentration Bendamustine HCl about 15 mg/mL Mannitol about25.5 mg/mL Alcohol about 10% (v/v) Water, q.s. to desired volume

Ingredients Concentration Bendamustine HCl about 2-40 mg/mL Mannitolabout 0-50 mg/mL Butanol about 0.5-20% (v/v) Water, q.s. to desiredvolume

Ingredients Concentration Bendamustine HCl about 5-20 mg/mL Mannitolabout 10-30 mg/mL Butanol about 1-10% (v/v) Water, q.s. to desiredvolume

Ingredients Concentration Bendamustine HCl about 12-17 mg/mL Mannitolabout 20-30 mg/mL Butanol about 1-10% (v/v) Water, q.s. to desiredvolume

Ingredients Concentration Bendamustine HCl about 15 mg/mL Mannitol about25.5 mg/mL Butanol about 10% (v/v) Water, q.s. to desired volume

Ingredients Concentration Bendamustine HCl about 2-50 mg/mL Mannitolabout 0-50 mg/mL Tertiary butanol about 0.5-100% (v/v) Water, q.s. todesired volume

Ingredients Concentration Bendamustine HCl about 2-50 mg/mL Mannitolabout 0-50 mg/mL Tertiary butanol about 0.5-99.9% (v/v) Water, q.s. todesired volume

Ingredients Concentration Bendamustine HCl about 2-50 mg/mL Mannitolabout 0-50 mg/mL Tertiary butanol about 0.5-99% (v/v) Water, q.s. todesired volume

Ingredients Concentration Bendamustine HCl about 2-50 mg/mL Mannitolabout 0-50 mg/mL Tertiary butanol about 90-99% (v/v) Water, q.s. todesired volume

Ingredients Concentration Bendamustine HCl about 5-20 mg/mL Mannitolabout 10-30 mg/mL Tertiary butanol about 5-80% (v/v) Water, q.s. todesired volume

Ingredients Concentration Bendamustine HCl about 12-17 mg/mL Mannitolabout 20-30 mg/mL Tertiary butanol about 10-50% (v/v) Water, q.s. todesired volume

Ingredients Concentration Bendamustine HCl about 12.5-15 mg/mL Mannitolabout 0-30 mg/mL Ethanol about 20-30% (v/v) Water, q.s. to desiredvolume

Ingredients Concentration Bendamustine HCl about 15 mg/mL Mannitol about25.5 mg/mL Tertiary butanol about 30% (v/v) Water, q.s. to desiredvolume

EXAMPLES

The following Examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in practicing theinvention. These Examples are in no way to be considered to limit thescope of the invention in any manner.

Materials:

Bendamustine HCl, (Degussa, Lot #s 0206005 and 0206007)

Mannitol, NF or equivalent (Mallinckrodt)

Ethyl Alcohol Dehydrated (200 proof), USP or equivalent (Spectrum)

Tertiary-butyl alcohol, ACS (EM Science)

Methanol (Spectrum and EMD)

Propanol (Spectrum)

Iso-propanol (Spectrum)

Butanol (Spectrum)

Water, HPLC grade or equivalent (EMD)

Acetonitrile, HPLC grade or equivalent (EMD)

Trifluoroacetic Acid, J. T. Baker

Methanol, HPLC grade or equivalent (EM Science, Cat #MX0488P-1)

Trifluoroacetic Acid, HPLC grade or equivalent (JT Baker, Cat#JT9470-01)

Equipment:

Waters 2695 Alliance HPLC system with photodiode array detector

Waters 2795 Alliance HPLC system with dual wavelength detector

Analytical Balance (Mettler AG285, ID #1028) and (Mettler X5205)

VirTis Lyophilizer AdVantage

Agilent Zorbax SB-C18 5 μm 80 Å 4.6×250 mm column, Cat#880975-902

Example 1 HPLC Procedures

Method 1

Mobile Phase A: 0.1% TFA; H₂O Mobile Phase B: 0.1% TFA; 50% ACN:50% H₂OUV: 230 nm Flow rate: 1.0 mL/min Column temp.: 30° C. Column: ZorbaxSB-C18 5 μm 80 Å 4.6 × 250 mm Sample temp.:  5° C. Injection Volume: 10μL Sample Concentration: 0.25 mg/mL in MeOH Gradient: 20% B for 1 min20-90% B in 23 min 90% B for 6 min back to 20% B in 1 min hold at 20% Bfor 4 min Run time: 30 min Post run time:  5 minMethod 2

Mobile Phase A: 0.1% TFA; H₂O:ACN (9:1) Mobile Phase B: 0.1% TFA;H₂O:ACN (5:5) UV: 230 nm Flow rate: 1.0 mL/min Column: Zorbax SB-C18 5μm 80 Å 4.6 × 250 mm Column temp.: 30° C. Sample temp.:  5° C. InjectionVolume: 10 μL Sample Concentration: 0.25 mg/mL in MeOH Gradient: 0% Bfor 3 min 0-50% B in 13 min 50-70% B in 17 min 70-90% B in 2 min 90% Bfor 5 min back to 0% B in 1 min hold at 0% B for 4 min Run time: 40 minPost run time:  5 minMethod 3

Phase A: HPLC grade water with 0.1% TFA(v/v) Phase B: HPLC gradeACN/water (1:1 v/v) with 0.1% TFA (v/v) UV: 254 nm Flow rate: 1.0 mL/minColumn: Zorbax SB-C18 5 μm 80 Å 4.6 × 250 mm Column temp.: 30° C. Sampletemp.:  5° C. Injection Volume: 5 μL Acquisition time: 30 min Post time: 9 min Diluent: methanolGradient:

Time (min.) % Phase A % Phase B 0.0 82 18 7.0 60 40 11.0 60 40 15.0 2080 30.0 20 80 31.0 82 18

Sample preparation—dissolve the drug product with 200 mL MeOH. Sonicate6 minutes. The solution can be injected directly into the HPLC (ca. 0.5mg/mL)

Method 4

Phase A: HPLC grade water with 0.1% TFA (v/v)

Phase B: HPLC grade ACN with 0.1% TFA (v/v)

UV: 254 nm

Flow rate: 1.0 mL/min

Column: Zorbax Bonus RP-C14 5 μm 4.6×150 mm

Column temp.: 30° C.

Sample temp.: 5° C.

Injection Volume: 2 μL

Acquisition time: 31 min

Post time: 5 min

Diluent: NMP/0.1% TFA in water (50:50 v/v)

Gradient:

Time (min.) % Phase A % Phase B 0.0 93 7 5 93 7 13 73 27 16 73 27 25 1090 31 10 90

Sample preparation for method 4—dissolve the drug product with a knownamount of diluent to prepare a concentration of 4.2 mg/mL for injectiondirectly into the HPLC. It may be necessary to perform a second dilution(the 100 mg/vial dosage form) to obtain a 4.2 mg/mL sampleconcentration.

Results

The retention times for some Bendamustine impurities using HPLC Method 1described above are shown in Table 11. An HPLC chromatograph forRibomustin® using the HPLC procedure described herein is shown in FIG.6.

TABLE 11 Retention Time for Bendamustine and some of its Impuritiesusing HPLC Method 1 Sample Name Retention Time (min) HP1 14.110Bendamustine 22.182 BM1 Dimer 24.824 BM1EE 26.968

Although HPLC Method 1 was capable of resolving impurities found inbendamustine it was not capable of separating a potential impurityformed during analysis, the methyl ester of bendamustine (BM1ME). Theretention time difference between BM1ME and BM1 Dimer was only 0.3minutes. In order to resolve BM1 Dimer, another HPLC method (#2) wasdeveloped. HPLC method #2 was capable of separating all the impuritiesbut required a longer run time of 45 minutes (Table 12).

TABLE 12 Retention Time for bendamustine and impurities using HPLCMethod 2 Sample Name Retention Time (min) HP1 15.694 BM1 25.420 BM1ME31.065 BM1 Dimer 32.467 BM1EE 36.038

The impurity profile of various lots of Ribomustin using HPLC Method 3are shown in Table 13.

TABLE 13 Ribomustine Impuirty Profile using HPLC Method 3 % Area BatchBendamustine(HCl) HP1 BM1EE BM1 Dimer BM1DCE 03H08 98.14 1.07 0.21 0.340.03 03H07 97.67 1.5 0.2 0.33 0.04 02K27 96.93 0.93 0.29 1.18 0.08 03C0897.61 1.24 0.19 0.46 0.02

Example 2 Solubility

The solubility of bendamustine HCl (bendamustine) in water (alone) andwith varying amounts of methanol, ethanol, propanol, isopropanol,butanol and tertiary-butyl alcohol (TBA) was determined by visualinspection. Amounts of bendamustine at 15 mg/mL, mannitol at 25.5 mg/mLwere prepared in 10 mL of the indicated alcohol solutions (Table 1) atroom temperature. Samples were then refrigerated at 5° C. and inspectedafter 0, 3, 6 and 24 hours for particulates and/or precipitates.

Results summarized in Table 1 indicate that bendamustine solubility isdependant on temperature and the amount of alcohol in aqueous solutions.For all alcohols the solubility of bendamustine increased as theconcentration of alcohol increased. The formation of a precipitant wasalso dependent on the temperature and time.

The solubility of bendamustine was also determined in 20% (v/v) TBAcontaining 25.5 mg/mL mannitol in water, and 30% (v/v) TBA containing25.5 mg/mL mannitol in water (FIG. 1). Bendamustine was added to 4 mL ofeach solution while mixing until it would no longer dissolve. Thesaturated solutions were allowed to mix for 1 hour at −8° C., 0° C., 5°C., or 25° C. The samples were centrifuged and placed back at theoriginal temperature for a minimum of 30 minutes. The −8° C. sample wasplaced into an ice bath containing sodium chloride, which lowers thetemperature of the ice bath, and the temperature was measured when thesample was pulled for analysis. An aliquot of each sample was taken andprepared for HPLC analysis.

The results of these experiments are shown in FIG. 1 and Table 2. Theamount of TBA, 20% (v/v) and 30% (v/v), used in the experiment (FIG. 1)was based on stability studies described herein.

As indicated in FIG. 1, the solubility of bendamustine decreasedlinearly with temperature (25° C. to 0° C.). The solubility ofbendamustine was temperature dependant whether it was dissolved in wateralone or with an alcohol. The 20% (v/v) TBA may likely be the lowerlimit required for efficient and robust pharmaceutical manufacturing dueto the stability and solubility of bendamustine. A filling solution of15 mg/mL bendamustine is close to the saturation limit of 17.2 mg/mLbendamustine at 5° C. but higher than the limit at 0° C. The 30% (v/v)TBA is the recommended concentration of TBA for the final formulationand is well within the solubility limit regardless of temperature.

Example 3 Stability

A. Stability in Water

Solutions of bendamustine (15 mg/mL), and mannitol (25.5 mg/mL) wereprepared in water at room temperature and immediately placed in an icebath (to lower the temperature quickly to about 5° C.) for 10 minutesand then refrigerated at 5° C. A sample of each formulation was analyzedby HPLC using the methods described herein after 0, 3, 6 and 24 hourswhen stored at 5° C.

B. Stability in Alcohols

Solutions containing 15 mg/mL bendamustine, 25.5 mg/mL mannitol, and1.9%, 5%, 10%, 20% or 30% (v/v) ethyl alcohol in water or 5%, 10%, 20%or 30% (v/v) TBA, methanol, propanol, iso-propanol, or butanol in waterwere prepared at room temperature, placed into an ice bath for 10minutes and then refrigerated at 5° C. A sample of each formulation wasanalyzed by HPLC after 0, 3, 6 and 24 hours when stored at 5° C.

C. Stability Results

Table 3 shows the stability results of bendamustine in water with noaddition of alcohol over a 24 hour period at 5° C. Bendamustine degradesquickly in water but the stability of bendamustine increases withincreasing alcohol concentrations (FIGS. 2, 3 and 4). Although alcoholsare frequently used in lyophilization to aid in solubility problems, theeffect of alcohols on bendamustine stability is unique, unexpected anduseful in manufacturing bendamustine with fewer impurities since anaqueous solution can be used while maintaining the stability ofbendamustine. TBA was found to be the best stabilizer of the sixalcohols tested (FIGS. 2, 3, and 4). All alcohols at 30% (v/v) reducedthe formation of impurities HP1 and Dimer at 5° C. for up to 24 hours.With respect to TBA, HP1 reaches only about 0.4% when stored at 5° C.for up to 24 hours. Lower concentrations of alcohol may not beefficient, when formulated at 15 mg/mL bendamustine and stored at 5° C.due to bendamustine precipitation and impurity formation.

Example 4 Formulation Optimization

After the solubility and stability of bendamustine were determined, theformulation was optimized for lyophilization. Since the concentration ofbendamustine is higher in a 30% TBA/water saturated solution as comparedwith other alcohol solutions, it is anticipated that the vial sizerequired to fill 100 mg of bendamustine can be decreased from thecurrent Ribomustin® presentation. Although a saturated solution ofbendamustine contains 18 mg/mL at 0° C., a concentration of 15 mg/mL wasselected for the formulation to compensate for slight differences in APIsolubility due to differences in bulk API purity as a result of batchdifferences. A concentration of 15 mg/mL bendamustine requires 6.67 mLto fill 100 mg of bendamustine HCl per vial.

The surface (sublimation) area to volume ratio is critical to producinga lyophilized product with good appearance that freeze dries quickly.Generally, lyophilized products occupy between 30% to 50% of the vialvolume. A 20 mL vial with 6.67 mL contains about 30% of its capacity andhas a surface area ratio of 0.796 cm²/mL.

Mannitol was selected as the bulking agent in order to maintain aformulation similar to Ribomustin®. Studies were performed to evaluatethe effect of mannitol on bendamustine solubility and appearance of theproduct. Mannitol decreases the solubility of bendamustine (at 15 mg/mL)in both ethanol and TBA aqueous solutions. For example, solutionscontaining 5% and 10% ethanol and TBA without mannitol did notprecipitate over 24 hours. However, for samples with mannitol (Table 1)precipitate was observed within 24 hours. There was no precipitate withaqueous solutions containing 30% (v/v) TBA, 15 mg/mL bendamustine, and25.5 mg/mL mannitol. In order to maintain a well formed cake resistantto breakage during handling, a minimum of 134 mg/vial of mannitol wasrequired with no difference observed in vials up to 200 mg/vial ofmannitol.

All alcohols tested increased the stability and solubility ofbendamustine. However, a significant mole fraction was required toaffect the stability of the filling solution and the ease ofmanufacturing. Smaller alcohols have the undesirable effect of loweringthe freezing point of the bulk solution and thus requiring longlyophilization cycles at lower temperatures. Higher concentrations ofmethanol and ethanol produced unattractive cakes that were difficult toreconstitute. 10% ethanol, 20% ethanol, 10% iso-propanol, 20%iso-propanol, or 30% TBA aqueous solutions containing bendamustine (15mg/mL), mannitol (25.5 mg/mL) were prepared and lyophilized. Thelyophilized vials filled from solutions of 10% ethanol, 20% ethanol, 10%iso-propanol, 20% iso-propanol produced either a collapsed cake or afilm residue. The only solvent system producing an acceptable cake was30% TBA. Additionally, reconstitution of 10% ethanol, 20% ethanol, 10%iso-propanol, 20% iso-propanol lyophilized vials were difficult and didnot fully dissolve until >45 minutes.

The ability to utilize a smaller vial is constrained by theconcentration or solubility of bendamustine in the aqueous/organicsolution. At lower concentrations of ethanol, methanol, isopropanol andn-propanol, which produced acceptable cake appearance, a more dilutesolution of bendamustine is required due to solubility limitations. Tomaintain a presentation with 100 mg of bendamustine per vial, a viallarger than 50 mL would be required. Also, stability studies hereinindicated that at the lower alcohol concentration, the chemicalstability was not sufficient to allow for acceptable filling times.

One of the factors affecting the ease of reconstitution is the porosityof the lyophilate. In general, amorphously precipitated solids withlittle surface area are more difficult to solubilize. Most lyophilatescontaining mannitol will reconstitute within 3-5 minutes as long asthere is no precipitate formed during lyophilization, frequently causedby evaporation of a liquid (melt back). Based on our experience withseveral lyophilization solvent systems and not wishing to be bound toany particular theory, the problems associated with Ribomustin®reconstitution may be associated with precipitation caused by melt backduring lyophilization. Most organic solvents do not lyophilizeefficiently and cause melt back because of their low melting point. TBA(tertiary butyl alcohol) has a high melting point and a similar vaporpressure as compared to water. TBA is removed by sublimation, notevaporation, at about the same rate as water. Lyophilates produced with30% (v/v) TBA according to the invention reconstitute within 3-10minutes as compare to commercially available Ribomustin which may take30-45 minutes.

Based upon the solubility, stability, ease of reconstitution andmanufacturing considerations, the following is a preferredpre-lyophilization formulation of the present invention: bendamustineHCl about 15 mg/mL, mannitol about 25.5 mg/mL, about 30% (v/v)tertiary-butyl alcohol, and q.s. using water for Injection. Theformulation is then filled at 5° C. using 6.67 mL in an amber 20 mL, 20mm vial and partially stoppered with a bromobutyl stopper and loadedinto a pre-chilled lyophilizer.

Example 5 Impurity Assessment

Major impurities introduced during Ribomustin® manufacturing,compounding, fill, and lyophilization procedure, as determined by HPLCanalysis (FIG. 6), are the hydrolysis product HP1, the Dimer, and theethyl ester of bendamustine, BM1EE. BM1EE can be formed during drugsubstance manufacturing, e.g., during recrystallization and/orpurification processes. BM1EE is known to be a more potent cytotoxicdrug than bendamustine. Experiments were undertaken to determine if theuse of a 30% TBA aqueous filling solution would lead to the formation ofbendamustine t-butyl ester.

Experiments were performed using traditional Fisher esterificationreaction conditions required for the formation of t-butyl ester ofbendamustine. Bendamustine was heated in 60° C. TBA with HCl for 20hours. No reaction was observed. This result indicated that it would bevery difficult to form the tert-butyl ester of bendamustine during thefill/finish process. No new impurities in drug product manufactured fromTBA have been observed in stability studies to date.

To aid in the testing of the drug product, synthetic routes using morereactive sources of the t-butyl moiety were developed. Another attemptto make tert-butyl ester was carried out by formation of the acylchloride of bendamustine. A suspension of bendamustine in methylenechloride was treated with oxalyl chloride and N,N-dimethylformamide.After acyl chloride was formed, the solvent was concentrated. Theresidue was added to methylene chloride, tert-butanol, triethylamine,and 4-dimethylaminopyridine and the mixture was stirred at roomtemperature overnight. After adding all solvents and purification, anunknown compound was given. The LC-MS did not match the molecular weightof bendamustine tert-butyl ester and the proton NMR did not showed thepeak for tert-butyl. Therefore, this attempt also failed to produce thebendamustine tert-butyl ester. Thus, using TBA as the co-solvent has anadditional benefit of not forming the ester from the alcohol.

Example 6 Lyophilization Cycle Development

Numerous lyophilization cycles were performed to evaluate the criticalstages of lyophilization and achieve the most efficient drying cycle.Experiments were performed to evaluate the effect of the freezing rate,primary drying temperature, time, and pressure on the product.

A. Freezing Rate

The literature reports that TBA adopts different crystal forms dependingon the freeze rate. In some TBA solutions, the slower the product froze,the quicker it dried. Larger crystals formed during slow freezingproducing bigger pores allowing more efficient sublimation. However,during studies with bendamustine, the freezing rate was not found to bea critical processing parameter when evaluated at 2 and 8 hours.

B. Primary and Secondary Drying

During the first attempts to lyophilize from 30% TBA solutions, thelyophilized cake fractured and powder was ejected from the vial. Thesecakes appeared to contain amorphous particles within the lyophilate, anindication of melt back. This phenomenon was reproducible and occurredwhen the product reached about −10° C. (refer to FIG. 5) independent ofthe warming rate. Several variables were tested to determine the causeand solution to the problem of the powder ejection. The pressure wasraised from 50 μm to 150 μm during primary drying, but powder ejectionwas still observed but to a lesser extent. This experiment was thenrepeated except the freezing rate was extended to 8 hours from 2 hours.This change had no effect.

The length of primary drying was next evaluated. For example, thefollowing very slow drying cycle was evaluated: freezing from +25° C. to−50° C. in eight hours; holding at −50° C. for 5 hours, warming anddrying from −50° C. to −25° C. in seven hours; holding for twenty hoursat −25° C., warming and drying from −25° C. to −15° C. in two hours andholding for twenty hours at −15° C., warming and drying from −15° C. to40° C. in six hours and holding for twenty hours at 40° C. whilemaintaining a chamber pressure of 150 μm throughout drying. No powderejection (FIG. 5) was observed. This cycle resulted in a well-formedcake without fracture that reconstituted readily. Without wishing to bebound to a particular theory, the problems with powder ejection anddifficulty with reconstitution may be the result of drying thelyophilate too quickly, thus resulting in strong vapor flow out of thecake as well as melt back. With the use of a less aggressive dryingcycle an aesthetic, stable, and easy to reconstitute cake wasreproducibly formed. Thus, removing all unbound water and tertiary-butylalcohol prior to secondary drying may prevent melt back as well aspowder ejection. The lyophilization cycle was further optimized underthese gentle conditions (FIG. 5). There were no immediate degradationproducts as a result of drying at 40° C. for up to 20 hours.

Example 7 Lyophilization Cycle

Pressure Step Description Time (Hour) Temperature (° C.) (Microns) 1Hold 0.25 5° C. — 2 Ramp 8 −50° C. — 3 Hold 4 −50° C. — 4 Ramp 3 −20° C.150 5 Hold 6 −20° C. 150 6 Ramp 1 −15° C. 150 7 Hold 20 −15° C. 150 8Ramp 0.5 −12° C. 150 9 Hold 15.5 −12 C. 150 10 Ramp 15 35 C. 50 11 Hold10 35° C. 50 12 Ramp 1 40 C. 50 Hold 5 40 C. 50 Total 89.25 — —

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the spirit and scopeof the invention. More specifically, it will be apparent that certainsolvents which are both chemically and physiologically related to thesolvents disclosed herein may be substituted for the solvents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit and scope of the invention asdefined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications are herein incorporated by reference tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed is:
 1. A vial containing a reconstituted solution ofbendamustine hydrochloride and mannitol in sterile water for injection,wherein the ratio by weight of bendamustine hydrochloride to mannitol inthe vial is 15:25.5, and wherein the bendamustine hydrochloride ispresent in the vial at a concentration of 100 mg per 20 mL.
 2. The vialof claim 1, wherein the vial contains 25 mg of bendamustinehydrochloride.
 3. The vial of claim 1, wherein the vial contains 100 mgof bendamustine hydrochloride.
 4. A 20 mL vial containing 100 mg ofbendamustine hydrochloride and 170 mg of mannitol reconstituted insterile water for injection.